Cell Lysis Solutions

Cell lysis solutions are detergent-based, buffers and reagent sets that have been optimized for particular cell lysis applications. Effective cell lysis and protein extraction for different species of organisms and different cell and tissue types require different buffer formulations. This article briefly describes available reagents for whole cell lysis and protein extraction. For a discussion of cell fractionation and organelle isolation, see the link in the sidebar.

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Cell lysis using detergents

Detergent cell lysis is a milder and easier alternative to physical disruption of cell membranes, although it is often used in conjunction with homogenization and mechanical grinding. Detergents break the lipid barrier surrounding cells by solubilizing proteins and disrupting lipid:lipid, protein:protein and protein:lipid interactions. Detergents, like lipids, self associate and bind to hydrophobic surfaces. They are comprised of a polar hydrophilic head group and a nonpolar hydrophobic tail and are categorized by the nature of the head group as either ionic (cationic or anionic), nonionic or zwitterionic. Their behavior depends on the properties of the head group and tail.

Unfortunately, there is no standard protocol available for selecting a detergent to use for membrane lysis. In general, nonionic and zwitterionic detergents are milder and less denaturing than ionic detergents and are used to solubilize membrane proteins where it is critical to maintain protein function and/or retain native protein:protein interactions for enzyme assays or immunoassays. CHAPS, a zwitterionic detergent, and the Triton-X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function.

The choice of detergent for cell lysis also depends on sample type. Animal cells, bacteria and yeast all have differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of the dense and complex nature of animal tissues, they require both detergent and mechanical lysis. In addition to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, salt concentration and temperature. Consideration should be given to the compatibility of the chosen detergent with downstream applications. If the detergent used for lysis must be removed, then a dialyzable detergent should be selected.

RIPA buffer is a popular choice for lysis and protein extraction of mammalian cells or soft tissue. Originally named after the assay method for which it was developed (radio-immunoprecipitation assay), RIPA buffer is effective when the immediate downstream application is SDS-PAGE (denaturing polyacrylamide gel electrophoresis). The formulation includes two ionic detergents and one nonionic detergent in Tris buffer: 25mM Tris•HCl, pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS).

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When the immediate downstream application for a lysate is a protein affinity purification experiment such as immunoprecipitation (IP) or co-immunoprecipiation (Co-IP), it is especially important to ensure that the lysis reagent does not contain denaturants or components that might interfere with the antibody binding or protein interactions of interest. RIPA buffer, despite its name, is not always the best choice for immunoprecipitation experiments because it contains SDS. Our M-PER Buffer (see next) is one alternative. Another option is to formulate your own lysis buffer or to test alternative commercial solutions to find the one that provides the best balance between extraction yield and IP success. Pierce IP Lysis Buffer was formulated to provide good results for IP overall.

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M-PER Mammalian Protein Extraction Reagent was developed as an effective yet milder alternative to RIPA buffer. M-PER Reagent uses a non-denaturing detergent to prepare total cell lysate that is compatible with many downstream assays including immunoassays, enzyme assays and a variety of common reporter assays. Lysis can be performed directly in culture plates and is completed in only 5 minutes. Furthermore, significantly more protein can be obtained with this method compared with freeze/thaw and sonication.

Comparison of M-PER Reagent with freeze/thaw cycles, sonication and Brand P lysis buffer. COS-7 cells grown in 100 mm plates at full confluency were washed once with 10mL of PBS, scraped with 1mL of PBS and centrifuged at 5000 rpm for 5 minutes to collect the cells. The cell pellets were resuspended in 0.5mL of respective extraction reagents and subjected to total protein extraction. For freeze/thaw cycles, the cell suspension in PBS was frozen in a dry ice and isopropanol bath for 10 minutes and thawed in a 37°C water bath. The freeze/thaw cycle was repeated three times. For sonication, the cell suspension was sonicated for 2 minutes with a 50% pulse using a Branson Sonifier 450 Sonicator. For extraction with M-PER Reagent and Brand P lysis buffer, the cell suspensions were shaken for 5 minutes. The cell debris was removed by centrifugation at 13,000 rpm for 5 minutes and the supernatants were assayed for protein concentration by the BCA Method.

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T-PER Tissue Protein Extraction Reagent is designed for total protein extraction from mammalian tissue samples, including heart, liver, kidney and brain. The T-PER Reagent utilizes a non-denaturing detergent in 25mM Bicine, 150mM NaCl, pH 7.6 and is used in conjunction with mechanical or manual homogenization. The resulting cell lysate, like the lysate prepared with M-PER Reagent, is compatible with many functional assays.

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B-PER Bacterial Protein Extraction Reagents gently lyse E. coli and other species of bacterial cells and effectively extract soluble native and recombinant proteins. B-PER Reagents have been used for Gram(-) bacteria, S. aureus, H. pylori and E. coli strains BL21(D3), JM109, DH5a and M15. The reagent is also suitable for certain Archaebacteria species and cultured insect cells. Extraction does not require expensive equipment and can be performed in less than 10 minutes. B-PER Reagent removes soluble protein from inclusion bodies and can be used to purify intact inclusion bodies whose less soluble proteins can be extracted by other means.

Several different ready-to-use formulations of B-PER Reagent are available for different lysis needs. These include formulations in Tris buffer or PBS, and those with and without Lysozyme and DNase I enzymes. The B-PER Direct Formulation is optimized for direct (homogenous) lysis of cells in 96-well culture plates, facilitating high throughput screening assays.

Two formulations have been developed. Y-PER Reagent is high salt (>300mM) and is effective for S. cerevisiae, S. pombe, C. albicans and P. pastoris (as well as various Gram-positive and Gram-negative bacteria). Although the detergent is compatible with various downstream methods, it is not dialyzable so cannot be removed in those cases where incompatibility exists. Y-PER Plus Dialyzable Reagent, (Part No. 78999) is a phosphate-free, low-salt formulation with a dialyzable detergent. It is validated for use primarily with S. cerevisiae.